An innovative platform to measure the activity of the sphingolipid pathway in single cells from primary, human, acute myeloid leukemia (AML) will be developed. A multidisciplinary group (chemist, bioengineer, oncologist and computational scientist) with a history of successful collaborations will pursue the development of engineered microfluidic instrumentation and supporting hardware using medically relevant probes to answer fundamental questions regarding heterogeneity in single AML cells. Fluorescent probes to track simultaneously the three major pathways comprising the ceramide-sphingosine axis in AML cells will be developed so that a detailed understanding of sphingolipid signaling in the tumor cells is achieved. Electrophoretic separations within a microfabricated device will be optimized for the single-cell measurements as a component of the work flow. Automation and integration will greatly increase throughput to yield a microdevice which is compatible with common clinical workflows. A powerful attribute of the proposal is that these measurements will be performed on single cells from primary samples and will avoid the confounding aspects of population- averaged data yielded by bulk cell assays. Furthermore, by simultaneously tracking all arms of the sphingolipid pathway, we will identify the strategies that AML cells use to dynamically reprogram their growth-promoting pathways via sphingolipid signaling during drug treatment. The proposed microfabricated devices will in the future provide key information concerning the best treatment option(s) for patients as well yielding an assessment of treatment efficacy to contribute fundamental data to the emerging field of precision medicine.